Although hepatitis C virus (HCV) is a leading cause of morbidity and mortality worldwide, the effects of viral gene expression on infected cells remain unclear in vivo. Efforts to develop culture and animal models for hepatitis C are critical for the understanding of the virus and the disease it causes. Our collaborators had previously shown that subgenomic replicon of the JFH-1 genotype 2a strain cloned from a patient with fulminant hepatitis replicates efficiently in cell culture. In a recent study, we showed that the full-length JFH-1 genome replicates efficiently and secrets viral particles after transfection into a human hepatoma cell line. The viral particles have a density of about 1.17 g/ml with an average diameter of about 55 nm. The secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies as well as immunoglobulins from chronically infected patients. The cell culture-generated HCV is infectious in chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing anti-viral strategies. Based on the tetracycline regulatory system, we established a binary transgenic model in which the conditional expression of two transgenes, SV40 T antigen (TAg) and LacZ, can be tightly regulated in the liver by administration of tetracycline. Mice with tTA and TAg transgenes developed hepatocellular adenomas and hyperplasia that could be prevented by continuous tetracycline administration. We have adopted this transgenic system to the regulated expression of HCV proteins in the liver using this model system. Analysis of Alb-tTA/TRE-HCV double transgenic mice revealed that the HCV RNA and protein expression in the liver could be completely suppressed by tetracycline administration, and induced in a reversible fashion by tetracycline withdrawal. Mice with constitutive expression of HCV had no evidence of hepatic pathology until 11 months of age, when steatosis developed. In contrast, mice with tetracycline-mediated suppression and then withdrawal at 2 months of age developed hepatic inflammation with ALT elevation by 4 months of age. Hepatic steatosis also became evident at 5 months of age, which occurred much earlier than the mice with constitutive HCV expression. Neither hepatic inflammation nor steatosis was observed in mice that had been continuously subjected to tetracycline treatment. With a tightly regulatable HCV expression, this model resembles those events observed in natural infection and offers a valuable tool to study the pathogenesis of hepatitis C. We have also initiated studies in chimpanzees, which are the only infectious animal model for hepatitis C. We have constructed a molecular infectious clone of HCV genotype 1b and studied the detailed virologic, serologic, and immunologic parameters of four chimpanzees inoculated with this monotypic inoculum. Two of the chimpanzees cleared the infection and two became persistently infected. Only weak and transient T helper responses were detected during the acute phase in all four chimpanzees. Liver biopsy samples collected over the course of infection were analyzed using cDNA microarray to characterize expression levels of a broad set of host genes. For three animals that exhibited a robust viremia (>10E5 genomes/ml) and evidence of liver injury, typical of acute HCV infection, the induction of a set of type I interferon regulated genes was observed during the first 8 weeks of infection, regardless of whether the animal eventually cleared the infection or not. However, the induction of certain genes related to signal transduction/cell growth could be associated specifically with viral clearance. During the viral clearance phase of one chimpanzee, intrahepatic activation of cellular immunity, particularly markers of CD8+ T-cells, was evident. On the other hand, the two chimpanzees that went on to chronic infection exhibited no evidence of such an adaptive immune response, despite the equally strong induction of the innate type I IFN response. These results are consistent with other reports that correlate in vitro hepatic CD8+ T-cell functions with clearance and they further support the notion that a vigorous intra-hepatic immune response is important for viral clearance. This observed difference possibly reside in the inability of the chimpanzee with chronic infection to activate the pathway transitioning from the innate to adaptive immunity. A fourth animal that exhibited a weak viremia (<500 genomes/ml) and quickly cleared infection (<8 weeks) had no detectable induction of type I interferon regulated genes or markers for T-cell response. Infection in this chimpanzee is consistent with a subclinical infection that probably occurs frequently in humans. This result suggests that HCV exposure can be controlled by an innate immunity, and if this process is effective during the initial phase of infection, adaptive immune response may not be necessary for viral clearance. These animal models are valuable not only to address issues of immunopathogenesis and cytopathic potential of HCV gene products but also to study HCV replication in vivo.